VCOM Louisiana Research Day Program Book 2024

Biomedical Research: Section 1

Kiara Pankratz, OMS-II; Annie Pham, OMS-II; Zachary Dickey, OMS-III; Elizabeth Kibodeaux, OMS-II; Erika Lytle, OMS-III; Dara W. Frank, PhD; Troy Stevens, PhD; Rebekah Morrow, PhD; K. Adam Morrow, PhD; Sarah Voth, PhD VCOM-Louisiana; Medical College of Wisconsin; University of South Alabama Whiddon College of Medicine 10 PSEUDOMONAS AERUGINOSA TOXIN EXOY CONSTRAINS LUNG ENDOTHELIAL AUTOPHAGIC FLUX TO PROMOTE TAU RELEASE

Background: The Gram-negative nosocomial pathogen Pseudomonas aeruginosa is the predominant agent responsible for nosocomial pneumonia and acute respiratory distress syndrome (ARDS). Patients frequently suffer poor physical and cognitive outcomes post discharge with less than 50% surviving the first year with that number following to one third in the following year. Over 90% of P. aeruginosa strain isolated from critically ill patients with acute nosocomial lung infection express a type III secretion system (T3SS) that works like a syringe-and-needle to inject a nucleotidyl cyclase toxin (ExoY) into the cell’s cytosol. ExoY generates unconstrained cyclic nucleotides within the host cell causing aberrant signaling and phosphorylation events. Intoxication of endothelium with ExoY at the air-blood barrier of host alveoli induces disruption of the endothelial barrier, alveolar flooding, and the production and release of hyperphosphorylated oligomeric tau into the extracellular milieu. ExoY generated endothelial derived tau is a heat-stable cytotoxin that can propagate from one cell to another and from one host to another with little loss of virulence. However, the mechanisms responsible for the generation of ExoY instigated pathogenic tau have not been resolved. Objective: We hypothesize that ExoY

constrains autophagic flux to promote the production and release of cytotoxic tau. Methods: Isogenic strains of P. aeruginosa expressing 1) an incompetent T3SS needle (∆PcrV; cannot form the translocation pore required to facilitate toxin injection), 2) an activity-null mutant unable to generate cyclic nucleotides in the host cell (ExoYK81M), and a mutant that injects only virulent ExoY (ExoY+) were used to infect rat pulmonary microvascular endothelial cells (PMVECs). Cells were treated with either vehicle or infected at a multiplicity of infection (MOI) of 20:1 in HBSS and incubated for 5 hours. ‘No cell’ wells of infection suspension only were incubated in parallel with infected cells as a control. Infected cells were imaged over time to assess signs of toxicity and endothelial barrier disruption. Supernatants and whole cell lysates were collected at 0 and 5 h post-infection and probed for LC3-I and LC3-II and endothelial derived tau harboring a phosphorylation-associated domain (PAD) using the TNT1 antibody. Lysate protein concentration was measured using a standard BCA assay. The antibody signal of lysates was normalized to GAPDH. TCA-DOC was used to precipitate supernatant samples. Samples of supernatant were loaded by volume and normalized to total protein. Cadaveric Alzheimer’s hippocampal

lysate and recombinant proteins were used as controls. Results: LC3-I identifies the autophagosome (cargo proteins ATG3/7). Lipidation of LC3-I produces LC3-II and signifies the fusion of the lysosome with the autophagosome (autolysosome). Vehicle treated cells exhibited the most robust LC3-I to LC3-II conversion/ turnover with greater LC3-II density. PMVECs infected with nonvirulent control strains ∆PcrV and ExoYK81M displayed equivalent levels of LC3-I to LC3-II conversion. ExoY intoxicated PMVECs displayed significantly reduced autophagic flux with a marked reduction in both LC3-I and LC3-II. The TNT1-reactive tau burden was largely ablated in supernatant fractions collected from ExoY+ infected cells. Moreover, pharmacologic activation of sirtuin 1 rescued autophagic flux while concurrently ablating PAD tau in the extracellular milieu. Conclusions: Our findings strongly implicate ExoY-mediated inhibition of endothelial autophagic flux as a mechanism of pathogenic tau production and release in nosocomial P. aeruginosa pneumonia.

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